The present invention relates generally to sample preparation, and more particularly to the preparation of crystallized samples used in scientific measurement instruments.
A source of uncertainty in scientific instrument measurements often lies in the preparation of samples. This is especially true in scientific instruments which use a crystallized analyte or a crystallized mixture of an analyte and a matrix material as a sample. Scientific instruments using crystallized samples perform functions such as mass spectrometry, laser desorption ionization, matrix assisted laser desorption ionization, ionization desorption achieved through fast atom bombardment, ionization desorption achieved through plasma desorption, ionization desorption achieved by electron impact or chemical ionization processes, and electron microscopy. All of these functions are well known to those skilled in the art.
To obtain accurate and reproducible measurements from these instruments, a homogeneous distribution of analyte crystals (or analyte/matrix co-crystals) must be produced on a sample surface. If the crystals are irregularly distributed, it is often necessary to view a magnified image of the sample surface to find a region of relatively evenly distributed crystals suitable for measurement.
Typically, an analyte or an analyte/matrix sample is prepared by first dissolving it into a homogeneous mixture using an aqueous or organic solvent. Such a solvent mixture is required to solvate matrix and analyte molecules, which may possess both hydrophobic and hydrophilic characteristics. Next, the liquid volatile components in a solvent/analyte mixture are removed through the application of heat or vacuum. As will be shown below, neither of these methods is ideal for the preparation of evenly distributed crystals on a sample surface.
For example, the application of heat to biological materials can degrade them, distorting any measurements taken of the biological analyte. Even when a heat tolerant analyte is used, rapid heating can cause boiling, which produces a number of mechanical, convective, and/or conductive perturbations on the sample surface. If the analyte dissolved in the organic solvent begins to crystallize before that which is dissolved in the aqueous, such perturbations can cause a shifting of position and differential deposition of the analyte crystals. The result can be an irregular distribution of analyte crystals upon the sample surface.
Less rapid heating also has its drawbacks when an analyte/matrix solvent mixture is used. If co-crystallization progresses slowly in such a mixture, solvent composition becomes increasingly hydrophilic as the more volatile, organic constituents vaporize. Accordingly, there is a time based crystallization order in which hydrophobic solutes crystalize before hydrophilic solutes. Since most matrix molecules are hydrophobic, they will preferentially co-crystalize with analyte solutes of low hydrophilicity. Thus, problems can occur when analyte solutes of high hydrophilicity are used.
When a vacuum is used to vaporize the liquid volatile solvent components in an analyte/solvent mixture (i.e., the opposing pressure is reduced so that it is less than the vapor pressure of the solvents involved), boiling also occurs. This produces the same mechanical, convective, and/or conductive perturbations on the sample surface as discussed above with respect to boiling caused by heating. Again, an irregular distribution of crystals on the sample surface often results.
An ultrasonic spray and rapid co-crystallization method of sample preparation using a variable vacuum is described in co-pending U.S. application Ser. No. 08/027,317, filed Mar. 4, 1993, and entitled Laser Desorption Ionization Mass Monitor. In this method, a layer of homogenous matrix/analyte solution is applied to a sample surface by an ultrasonic spray apparatus producing a very fine mist. The layer is then crystallized by applying a variable vacuum to the sample surface to remove volatile fluid components. The ability of an operator to view the sample surface and to vary the vacuum applied reduces the mechanical and other perturbations caused by boiling of the volatile liquid solvent components, and results in a more homogeneous distribution of crystals across the sample surface. While such a controlled process provides an improvement over previous sample preparation techniques, the mechanical perturbations inherent in any boiling technique are still present to some degree. Also, this method does not lend itself to automation since a human operator is required to view the sample and to control the vacuum pressure.
U.S. Pat. No. 5,045,694 discloses an electrospray method of applying an analyte/matrix mixture to a sample surface. Although this method appears to produce good quality analyte/matrix crystals, it involves applying a potential of about five thousand volts to the sample surface during application of the layers. This makes the method somewhat hazardous, and can lead to corona discharge between the sample surface and the spray apparatus. This may damage both the sample surface and spray apparatus.
Another method for sample preparation was described in an article entitled "Device for Controlling Crystallization of Protein," NASA Tech Briefs, Vol. 17, No. 9, Sep. 1993, pp. 92-93. In this method, a variable sandwich spacer enables the optimization of the evaporative driving force that governs the crystallization of a protein from solution. The method allows the growth of very large crystals, which are important for applications such as x-ray crystallography. However, this method is expensive since it requires a complex apparatus to be performed. Also, such large crystals are not required for most of the scientific measurement instruments described above.